Name: in vivo crispr/cas9 screening to simultaneously evaluate gene function in mouse skin and oral cavity
Uploaded: 2020-11-02T13:00:00
Description: Watch this Scientific Journal Video about In Vivo CRISPR/Cas9 Screening to Simultaneously Evaluate Gene Function in Mouse Skin and Oral Cavity at JoVE.com

这项工作得到了加拿大卫生研究所（CIHR 365252）、克雷姆比尔基金会和安大略研究基金卓越研究第8轮（RE08-065）的项目赠款的支持。桑帕德·库马尔·洛加坦是加拿大癌症协会奖学金（BC-F-16#31919）的接受者。

该协议是根据多伦多大学的IACUC批准和执行的。
1. 集合的CRISPR库的设计和克隆
<ol><li>选择 4-5 sgRNA，从资源中针对感兴趣的鼠标基因，如博德研究所 sgRNA 设计器 （https://portals.broadinstitute.org/gpp/public/analysis-tools/sgrna-design） 或 CHOPCHOP 服务器 （https://chopchop.cbu.uib.no）。从 Sanjana 等9 中选择同等数量的非目标 sgRNA，以生成同样大小的非目标控制 gRNA 库。</li>
	<li>?...

<ol>
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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrafast+and+memory-efficient+alignment+of+short+DNA+sequences+to+the+human+genome.">Ultrafast and memory-efficient alignment of short DNA sequences to the human genome.</a> Genome Biology. 10, 25 (2009).</li><li>Li, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MAGeCK+enables+robust+identification+of+essential+genes+from+genome-scale+CRISPR/Cas9+knockout+screens.">MAGeCK enables robust identification of essential genes from genome-scale CRISPR/Cas9 knockout screens.</a> Genome Biology. 15, 554 (2014).</li><li>Winters, I. P., Murray, C. W., Winslow, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+quantitative+and+multiplexed+in+vivo+functional+cancer+genomics.">Towards quantitative and multiplexed in vivo functional cancer genomics.</a> Nature Reviews Genetics. 19, 741-755 (2018).</li><li>Rogers, Z. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+the+in+vivo+fitness+landscape+of+lung+adenocarcinoma+tumor+suppression+in+mice.">Mapping the in vivo fitness landscape of lung adenocarcinoma tumor suppression in mice.</a> Nature Genetics. 50, 483-486 (2018).</li><li>Chow, R. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AAV-mediated+direct+in+vivo+CRISPR+screen+identifies+functional+suppressors+in+glioblastoma.">AAV-mediated direct in vivo CRISPR screen identifies functional suppressors in glioblastoma.</a> Nature Neuroscience. 20, 1329-1341 (2017).</li><li>Wang, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mapping+a+functional+cancer+genome+atlas+of+tumor+suppressors+in+mouse+liver+using+AAV-CRISPR&#8211;mediated+direct+in+vivo+screening.">Mapping a functional cancer genome atlas of tumor suppressors in mouse liver using AAV-CRISPR&#8211;mediated direct in vivo screening.</a> Science Advances. 4, 5508 (2018).</li><li>Chan, K., Tong, A. H. Y., Brown, K. 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CRISPR/Cas9基因组编辑已广泛应用于体外和体内研究，以研究基因功能和细胞过程。大多数体内研究都利用CRISPR/Cas9基因编辑的细胞移植到动物模型（异种移植或异种移植）。虽然这是研究癌症遗传学和细胞功能的有力工具，但它仍然缺乏原生组织微环境，并可能引起伤害和/或免疫反应。
为了克服这些挑战，几个小组已经率先在体内CRISPR方法产生几个，多路复用的自动鼠标模型...

后基因组时代癌症研究面临的挑战之一是挖掘因果基因突变的大量基因组数据，并识别基因网络中可以针对治疗的节点。虽然生物信息分析对实现这些目标大有帮助，但建立有效的体外和体内模型是破译生物系统和疾病状态的复杂性和促进药物开发的先决条件。虽然传统的转基因小鼠模型在体内癌症遗传学研究中被广泛使用，但其成本、时间和劳动密集型性质在很大程度上禁止了对现代基因组学解开的数百个假定癌症基因的系统分析。为了克服这一瓶颈，我们结合了先前建立的子宫内注射方法1，2的超声波引导与CRISPR/Cas9（聚集定期间歇短巴林德罗米重复）基因编辑技术3，同时诱导和研究数百个基因在皮肤和单只小鼠口腔的功能丧失突变。
此处描述的方法利用工程扁豆病毒的超声波引导注射到胚胎日E9.5活小鼠胚胎的羊水腔中。含有CRISPR/Cas9成分的扁桃体货物会转导单层表面等体，后者后来导致皮肤和口腔的上皮。皮肤由外表皮组成，其次是地下室膜和真皮。表皮是一种分层上皮，由内基层组成，与地下膜保持接触，具有增殖和干细胞能力。基底层产生上面的分化层，如旋转层、颗粒层和地层角膜层2、4。血统追踪研究表明，这种直接在体内CRISPR/Cas9方法基因操纵组织驻存干细胞在基底层，持续整个成年。由于扁豆病毒可以滴定以在克隆密度下转导E9.5表面细胞，因此这种方法可用于生成含有数千个离散基因克隆的马赛克小鼠。然后，下一代测序可用于以多路复用的方式分析CRISPR/Cas9介质基因消融在这些克隆中的影响。
我们最近使用这种方法评估了484个基因的功能，这些基因显示人类头部和颈部鳞状细胞癌（HNSCC）5的反复突变。HNSCC是一种毁灭性的癌症，死亡率高达40-50%，是全世界第6大最常见的癌症。HNSCC 出现在上气道或口腔的粘膜衬里中，与烟草和酒精消费或人类瘤病毒（ HPV ）感染有关。皮肤鳞状细胞癌（SCC）是皮肤肿瘤，是人类第二常见的癌症。角质SCC和HNSCC在病理学和分子学上非常相似，在TP53、PIK3CA、NOTCH1和HRAS8中出现改变的病例比例很高。虽然只有少数基因在高频下发生变异，但有数百个基因在低频下发生变异（+lt;5%），这种现象通常被称为长尾分布。由于大多数长尾基因缺乏生物或临床验证，我们利用这种体内CRISPR筛选技术，在p53、Pik3ca或Hras中具有敏感突变的肿瘤易发小鼠中模拟这些基因的功能丧失，并发现了几个与p53、Pik3ca或Hras合作的新型肿瘤抑制基因，以触发肿瘤发育5。
在这里，我们描述了一个详细的协议，以产生多路sgRNA扁平CRISPR sgRNA库，并在鼠标表面等体中执行CRISPR/Cas9基因敲除屏幕。值得注意的是，这种方法可以应用于整合其他基因操作技术，如CRISPR激活（CRISPRa）和CRISPR灭活（CRISPRi），或修改为针对小鼠的其他器官系统来研究基因功能。

<table>
	<tbody>
		<tr>
			<td>0.45 micron filter</td>
			<td>Sigma</td>
			<td>S2HVU02RE</td>
			<td></td>
		</tr>
		<tr>
			<td>12k or 92k oligo chip</td>
			<td>Customarray Inc. (Genscript)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>15 cm cell culture plates</td>
			<td>Corning</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>293FT</td>
			<td>Invitrogen</td>
			<td>R70007</td>
			<td></td>
		</tr>
		<tr>
			<td>293NT</td>
			<td>Systems Biosciences</td>
			<td>LV900A-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Alkaline phosphatase</td>
			<td>NEB</td>
			<td>M0290L</td>
			<td></td>
		</tr>
		<tr>
			<td>Amplicillin</td>
			<td>Fisher Scientific</td>
			<td>BP1760-25</td>
			<td></td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>NEB</td>
			<td>9804S</td>
			<td></td>
		</tr>
		<tr>
			<td>BsmBI</td>
			<td>NEB</td>
			<td>R0580L</td>
			<td></td>
		</tr>
		<tr>
			<td>Chromic gut suture</td>
			<td>Covidien</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Deep sequencing (Next-Seq or Hi-Seq)</td>
			<td>Illumina</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DNA-cleanup kit</td>
			<td>Zymo Research</td>
			<td>D4008</td>
			<td></td>
		</tr>
		<tr>
			<td>DNAesy Blood and Tissue DNA extraction kit</td>
			<td>Qiagen</td>
			<td>69506</td>
			<td></td>
		</tr>
		<tr>
			<td>Endura electrocompetent cells</td>
			<td>Lucigen</td>
			<td>60242-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass Capillaries</td>
			<td>Drummond</td>
			<td>3-000-203-G/X</td>
			<td></td>
		</tr>
		<tr>
			<td>HEK293T cells</td>
			<td>ATCC</td>
			<td>CRL-3216</td>
			<td></td>
		</tr>
		<tr>
			<td>High-Speed Centrifuge</td>
			<td>Beckman Coulter</td>
			<td>MLS-50</td>
			<td></td>
		</tr>
		<tr>
			<td>LB Agar</td>
			<td>Wisent Technologies</td>
			<td>800-011-LG</td>
			<td></td>
		</tr>
		<tr>
			<td>Micropipette puller</td>
			<td>Sutter Instrument</td>
			<td>P97</td>
			<td></td>
		</tr>
		<tr>
			<td>Mineral oil</td>
			<td>Sigma</td>
			<td>M5904</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini-prep plasmid Kit</td>
			<td>Frogga Bio</td>
			<td>PDH300</td>
			<td></td>
		</tr>
		<tr>
			<td>Mouse oxygen anaesthesia system</td>
			<td>Visual Sonics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nanoject II micromanipulator</td>
			<td>Drummond</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>NEBuffer 3.1 (Buffer for BsmBI)</td>
			<td>NEB</td>
			<td>R0580L</td>
			<td></td>
		</tr>
		<tr>
			<td>Needle sharpener</td>
			<td>Sutter Instrument</td>
			<td>BV-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Oligo cleanup kit</td>
			<td>Zymo research</td>
			<td>D4060</td>
			<td></td>
		</tr>
		<tr>
			<td>PAGE purified illumina sequencing primer</td>
			<td>IDT DNA</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>PEI (polyethyleneimine)</td>
			<td>Sigma</td>
			<td>408727-100ML</td>
			<td></td>
		</tr>
		<tr>
			<td>Permoplast modeling clay</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Petridish with central opening</td>
			<td>Visual Sonics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>pMD2.G</td>
			<td>Addgene</td>
			<td>12259</td>
			<td></td>
		</tr>
		<tr>
			<td>psPAX2</td>
			<td>Addgene</td>
			<td>12260</td>
			<td></td>
		</tr>
		<tr>
			<td>Q5 Polymerase 2x Master mix</td>
			<td>NEB</td>
			<td>M0494L</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit Fluorometric Quantification</td>
			<td>Invitrogen</td>
			<td>Q33327</td>
			<td></td>
		</tr>
		<tr>
			<td>Semicircular Silicone plug</td>
			<td>Corning</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Silicone membrane</td>
			<td>Visual Sonics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>T4 DNA ligase</td>
			<td>NEB</td>
			<td>M0202L</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultra-centrifuge tubes</td>
			<td>Beckman Coulter</td>
			<td>344058</td>
			<td></td>
		</tr>
		<tr>
			<td>Vevo2000 ultrasound system</td>
			<td>Visual Sonics</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

in vivo crispr/cas9 screening to simultaneously evaluate gene function in mouse skin and oral cavity

转基因小鼠模型 （GEMM） 在评估基因功能、模拟人类疾病以及作为临床前模型评估治疗途径方面发挥了重要作用。然而，它们的时间、劳动和成本密集型性质限制了它们对基因功能进行系统分析的效用。基因组编辑技术的最新进展克服了这些限制，并允许以多路复用和快速的方式在特定小鼠器官内快速生成特定的基因扰动。在这里，我们描述了一种基于CRISPR/Cas9的方法（聚集定期间歇短脑重复），在小鼠皮肤和口腔的上皮产生数千个基因敲除克隆，并提供一个协议，详细说明   在肿瘤抑制基因的直接体内CRISPR屏幕执行必要的步骤。这种方法可以应用于其他器官或其他CRISPR/Cas9技术，如CRISPR激活或CRISPR灭活，以研究基因在组织失落期间或在各种疾病环境中的生物功能。

图1A 显示了寡核苷酸的设计，用于在单个 12k 或 92k 寡头芯片中以经济高效的方式对多个自定义 CRISPR 库进行多路复用。一旦选择 sgRNA（蓝色编码），寡核苷酸将设计为限制位点（橙色 BsmBI）和库特定的 PCR 引物对（绿色编码）。几个库可以通过使用引物对的独特组合来设计，用于在单个寡头芯片中进行多重组合。当 PCR 使用特定的 PCR 底漆对放大库时，始终只包含水负控制，?...

Watch this Scientific Journal Video about In Vivo CRISPR/Cas9 Screening to Simultaneously Evaluate Gene Function in Mouse Skin and Oral Cavity at JoVE.com

In Vivo CRISPR/Cas9 Screening to Simultaneously Evaluate Gene Function in Mouse Skin and Oral Cavity

在这里，我们描述了一种快速和直接的体内CRISPR/Cas9筛查方法，使用超声波引导子宫胚胎扁平注射，同时评估几个基因在皮肤和免疫能力小鼠口腔的功能。

在Vivo CRISPR/Cas9筛查中同时评估小鼠皮肤和口腔中的基因功能

Genetically modified mouse models (GEMM) have been instrumental in assessing gene function, modeling human diseases, and serving as preclinical model to ...

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QTL Mapping and CRISPR/Cas9 Editing to Identify a Drug Resistance Gene in Toxoplasma gondii

QTL Mapping and CRISPR/Cas9 Editing to Identify a Drug Resistance Gene in Toxoplasma gondii

QTL定位和CRISPR / Cas9编辑识别抗药性基因<em&gt;弓形虫</em

Scientific knowledge is intrinsically linked to available technologies and methods. This article will present two methods that allowed for the ...

科研

遗传学

Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells

选择依赖和独立生成CRISPR / Cas9介导的哺乳动物细胞基因敲除

The CRISPR/Cas9 genome engineering system has revolutionized biology by allowing for precise genome editing with little effort. Guided by a single guide ...

Primordial Germ Cell Transplantation for CRISPR/Cas9-based Leapfrogging in Xenopus

Primordial Germ Cell Transplantation for CRISPR/Cas9-based Leapfrogging in Xenopus

CRISPR/Cas9-based 跳跃的原始生殖细胞移植在爪

The creation of mutant lines by genome editing is accelerating genetic analysis in many organisms. CRISPR/Cas9 methods have been adapted for use in the ...

A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells

CRISPR/Cas9 和 SsODN 在人细胞中催化点突变修复功能的标准方法研究

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point ...

Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing

Microinjection of CRISPR/Cas9 Protein into Channel Catfish, Ictalurus punctatus, Embryos for Gene Editing

CRISPR/Cas9 蛋白注射到沟鲶、鮰松毛虫、基因编辑的胚胎

The complete genome of the channel catfish, Ictalurus punctatus, has been sequenced, leading to greater opportunities for studying channel catfish gene ...

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

CRISPR/Cas9-mediated Targeted Integration In Vivo Using a Homology-mediated End Joining-based Strategy

As a promising genome editing platform, the CRISPR/Cas9 system has great potential for efficient genetic manipulation, especially for targeted integration ...

Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio

Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio

斑马斑马模型系统中 CRISPR/Cas9-generated 基因挖空的高效生产与鉴定

Characterization of the clustered, regularly interspaced, short, palindromic repeat (CRISPR) system of Streptococcus pyogenes has enabled the development ...

Highly Efficient Gene Disruption of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9

CRISPR/Cas9 对小鼠和人造血祖细胞的高效基因破坏

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. ...

CRISPR/Cas9 Gene Editing to Make Conditional Mutants of Human Malaria Parasite P. falciparum

CRISPR/Cas9 Gene Editing to Make Conditional Mutants of Human Malaria Parasite P. falciparum

CRISPR/Cas9 基因编辑制作人类疟疾寄生虫的条件突变体

Malaria is a significant cause of morbidity and mortality worldwide. This disease, which primarily affects those living in tropical and subtropical ...

CRISPR/Cas9 Ribonucleoprotein-mediated Precise Gene Editing by Tube Electroporation

CRISPR/Cas9 核糖核酸蛋白介导的精确基因编辑通过管电穿孔

Gene editing nucleases, represented by CRISPR-associated protein 9 (Cas9), are becoming mainstream tools in biomedical research. Successful delivery of ...